MYCOLOGY

Margie Morgan, PhD D(ABMM)

Just the basics – meant for board review or brief study of this fascinating area of microbiology!

Mycology

Starting point

Yeast are:• Unicellular / produce budding daughter cells• Colony on solid media are usually white to beige and

appear much like bacterial colonies• Some genera produce mucoid colonies

(Cryptococcus)

Starting point

Molds are:• Filamentous with hyphae• Produce conidia [spores]• Colonies on solid agar are downy, fluffy, cottony• Most mold colonies are pigmented which aid in

identification

hyphaespores

Appropriate Specimens and Transport conditions for Fungal disease diagnosis• Fungi are very hardy organisms • No requirement for special transport media for culture

submission• Sterile containers used to prevent bacterial contamination• Numerous sites are appropriate for culture• Respiratory specimens – sputum, bronchial lavage,

brushings, nasal sinuses• Tissue biopsies• Cutaneous - Skin scrapings, material from lesions• Ocular• Sterile body fluids including CSF• Blood, bone marrow

Fungal Culture Media• Sabouraud’s glucose agar (SABS)

All purpose Fungal media – nothing to discourage bacteria to grow

SABS best used for the subculture of fungi for workup

Contains 2% glucose, pH @7.0

• Inhibitory mold agar Selective SABS agar with chloramphenicol, gentamicin [inhibit bacterial growth] and enrichment,

Good for the primary recovery of dimorphic pathogenic fungi. Saprophytic fungi and dermatophytes inhibited

on inhibitory mold agar

Common Fungal Media• Mycosel/Mycobiotic agars

• Selective SABS with chloramphenicol and cycloheximide • Used for culture of dermatophytes – fungi that cause skin, hair and nail

infections• Brain heart infusion agar

• Primary recovery of all fungal organisms• Can make it more selective by adding chloramphenicol and

cycloheximide• Can add blood to agar to nurture systemic fungi

• Fungal cultures incubated for 4 weeks at 30˚C , room air• Lower temperature than bacterial culture incubation [35˚C]• If plates are used for fungal cultures the plates must be sealed with

air permeable tape for laboratory safety

What effect does Cycloheximide have when added to media?• Prevents rapidly growing molds from overgrowing dimorphics and dermatophytes• This is the positive aspect of cycloheximide in media

• Beware: it is not all good, it can suppress important fungi from growing. Inhibited fungi include:• Trichosporon beigelii• Candida tropicalis• Cryptococcus neoformans• Yeast phase of Blastomyces• Yeast phase of Histoplasma

Inoculate fungal media

Seal plates with tape to prevent culture contamination and escape of fungal spores

Incubate at 30˚C for 4 wk

If growth occurs - perform proper identification methods:

Yeast identification methods Manual and automated biochemical reactions capable of identifying most yeast species.

Newer methods [Mass spectrometry – MALDI-TOF and 16 sRNA sequencing

Processing of Fungal Cultures - stepwise

Lactophenol cotton blue [LCB] adhesive tape preparation is method used for mold identification.

LCB mounting medium consists of phenollactic acid, glycerol and aniline cotton blue dye.

Clear adhesive (scotch) tape touches a mold colony, picking up fungal hyphae and pressed into one drop of LCBon a microscope slide.

If LCB prep is not able to identify a mold 16sRNAsequencing can be usedto identify problematic molds in reference laboratories.

Mold Identification methods

Safety in the Mycology Laboratory• If a culture is growing a mold, it cannot be opened on the

bench top• All mold work must be performed in a BSL-2 biosafety

cabinet with Hepa filtration• Yeast identification can be performed on the bench top

Direct Exams used to identify fungi directly from patient specimens• Gram stain – all specimen types can be Gram stained.

Can only reliably detect yeast by Gram stain.

• KOH preparation – Skin, Hair or Nails examined for both yeast and/or hyphae

• Calcofluor white stain – all specimen types can be stained and examined for yeast and/or hyphae

• India ink – Primarily used for CSF for the detection of Cryptococcus neoformans and C. gattii

Yeast cells stain blue [Gram positive]. Examine for budding cells to confirm that it is a yeast cell and not an artifact. Examination on oil immersion lens.You can also detect pseudohyphae on Gram stain. Mold can be difficult to identify on a Gram stain.

pseudohyphae

moldpseudohyphae

Gram Stain

Used to detect yeast and/or hyphae in skin, hair and nail specimens using 40X light microscope.KOH dissolves keratin found in cell materialand frees hyphae from the cellKOH exams can be difficult to interpret!

KOH – potassium hydroxide prep

Yeast, pseudohyphae, and mycelial fungi bind with the Calcofluor white stain. Prep is interpreted using a fluorescence microscope.Sensitivity and specificity is improved over the KOH preparation.

Calcofluor white stain

One drop of black ink is placed into one drop of CSF and examined using a 40X lens on light microscope

It is a “negative” stain, staining the background not the yeast cellThe clearing is thepolysaccharide capsule ofCryptococcus neoformans or C. gattii. Specificity is improved if you look for budding yeast cells.

India Ink

Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black.

Examine the hyphae for presence of septations in the hyphae, broad or more narrow width and angle of branching.Examine the size and budding pattern of observed yeast.We will observe on later slides how these criteria can assist in identification.

Examination of fungi in fixed tissue

PAS-positive staining red against a green or blue background

Periodic Acid Schiff [PAS]

Stain-Cryptococcus neoformans polysaccharide capsule stains pink

Mucicarmine [Mucin] stain

Great for cellularity, but GMS, or PAS better show features of the fungi.

Hematoxylin and Eosin Stain

DIMORPHIC FUNGIImportant systemic pathogens with some unique characteristics

What does Dimorphic mean?

• Two forms exist for one fungus species depending on temperature and conditions of environment• Mycelial form - Hyphae and conidia

• free living form found in nature and at laboratory temperature <=30˚C• Yeast or yeast like form

• parasitic phase found in human tissue or in the lab >= 35˚

Histoplasma capsulatum – moldfrom 30˚C culture

Histoplasma capsulatum – yeastfrom tissue and 35˚C culture

Dimorphic Fungi capable of causing systemic infection – most common

• Histoplasma capsulatum• Blastomyces dermatitidis• Coccidioides immitis• Paracoccidioides brasiliensis• Sporothrix schenckii• Penicillium marneffei

Histoplasma capsulatum• World wide distribution / In USA in Ohio, Missouri, and Mississippi River valleys considered endemic

• Associate with Bat guano (Spelunker = cave explorers) and bird droppings

Histoplasmosis Disease• 95% of infections are subclinical• 5% infections:

• Progressive pulmonary• Chronic systemic infection with dissemination to the RES

system including bone marrow• Acute fulminating systemic disease (fatal)

• Reactivation disease can occur in elderly and immunosuppressed (AIDS is a good example)

• Bone marrow exam is useful in diagnosing disseminated infections

• Mucocutaneous lesions are a unique & common site of dissemination

The yeast of Histoplasma capsulatum prefer to be intracellular and inhabit

Macrophages.Small 2 – 4 um, regular in size, and oval to round.

Do not have a capsule, this is just a staining artifact.

H & E PAS

Gram Wright’s

Histoplasma will stain with a variety of stains

Histoplasmosis rapid diagnosis

• Antigen detection in urine• Quantitative Enzyme immunoassay• Random urine specimen• Most useful for disseminated infection and chronic

pulmonary disease• Antigen is detectable in >=85% of these infections• Good for diagnosis of immune suppressed patients

that do not produce a detectable antibody response• Antibody tests are available but have been replaced

mostly by Antigen detection

Histoplasma capsulatum• Fungal culture incubated at 30˚C

• Very SLOW growing taking 2 – 8 weeks to form colonies• Colony is white to brown and cottony • Microscopic appearance – tuberculated macroconidia that are

large and round (8 – 16 µM) plus small microconidia (2 - 4µM) [see picture]

• Microconidia are the infectious particle growing in nature and capable of penetrating deep into the lung

• DNA probe must be used to confirm identification so there is no confusion with look alike fungi to the inexperience

• Sepedonium species looks somewhat like Histoplasma and is considered a look a like fungus – it is a contaminate

Histoplasma capsulatum culture at 30˚C is white and cottony.

Microscopic exam: Tuberculate [projections]on the macroconidia.Microconidia are the infectious particle. Thin hyaline septate hyphae

Appearance in culture at 30 degrees C

Sepedonium species

Appearance in culture at35 degrees C

• Culture at 35˚C is yeast phase• Grow as small yeast, round to oval, always consistent

in size and shape (2 -4 uM) narrow neck at the budding juncture

Histoplasma capsulatum in tissue• Granulomas are usually produced and can be either

caseating or non caseating• Infection usually begins by breathing in the microconidia

and infecting the Lung• Infection disseminates to organs of the

Reticuloendothelial System (RES) – with high % of dissemination to the Bone Marrow

• Intracellular budding yeast (2 – 4 µM) are seen in all tissues

Leishmania speciesNote small round kinetoplast next to nucleus

Toxoplasma

Histoplasma capsulatum

Beware of look alike organisms in tissue specimens!!

H capsulatum var duboisii yeast cells are 8 – 10 uM in size, which is 2X the size of regular Histoplasma capsulatum yeast cells.

H. capsulatum var duboisii disease is found in Central AfricaCauses infection in skin and boneThe 30˚C culture is identical to H capsulatum.

Unusual variant of H. capsulatum

Blastomyces dermatitidis• Epidemiology

• Ohio and Mississippi River valleys• No association with specific animal or activity• Forrest and river banks?• Primarily a pulmonary infection which may disseminate to the skin

and bone

Well demarcated skin lesion is typicalScraping of skin lesions are full of yeast cells

Blastomyces dermatitidis• Culture at 30˚C

• Grows in 2- 3 weeks• Fluffy white – buff colored mold, prickly• Pear shaped conidia at the end of supporting hyphae

– looks like lollipops• Look alike fungus – Chrysosporium species• Do DNA probe test to confirm identification

BlastomycesChrysosporium

Slow growing yeast colony taking @ 4 weeks to form a colonyYeast cell is 8 – 20 um in size and is unique for it’s Broad Based Budding pattern and the double contoured wall.

Blastomyces culture at 37 degrees C

Blastomyces dermatitidis histopathologyMixed pyogenic and granulomatous inflammation is observed in tissue with Broad based budding yeast cells

Coccidioides immitis

• Endemic in SW USA, Mexico, South America, in areas known

as the Sonoran life zone with a warm climate and desert sandsInfection is from inhalation of fungal particles found in the sand

• Coccidioides posadasii is a genetically related to C. immitis. The two species are located in different endemic regions, but produce the same disease process

Coccidioidomycosis• 95% of infections are asymptomatic or with limited

symptoms • The remaining 5% are focal pulmonary, progressive

pulmonary or disseminated infections. Dissemination to the central nervous system is difficult to cure and has a high fatality rate.

• Higher incidence of dissemination occurs in patients with: • defects in cell mediated immunity (HIV), • darker skinned ethnic groups, • pregnancy

Coccidioides immitis [posadasii]

• Culture at 30˚C• Requires only 2 – 3 days to grow, colony starts waxy and

becomes wooly in around 7 – 10 days• Under the microscope one looks for foci of septated hyphae

with thick walled barrel shaped arthroconidia with clear spaces in between. The clear spaces are dead arthroconidia.

• Arthroconidia infectious particle in nature• Very infectious to laboratory personnel

Coccidioides • Malbranchea species can look like C. immitis under the

microscope• Because of look-a-like fungi one needs to confirm

identification of Coccidioides immitis with DNA probe or similar method to be sure!

Coccidioides Malbranchea

Barrel shaped alternating arthroconidia are produced in cultures grown at both 30 and 35 C.There is no yeast phase for C. immitis [posadasii]

No yeast phase with Coccidioides!

Coccidioides Histopathology• Thick walled spherules (10 – 80 uM) with endospores

are seen in tissue. This is the second form of Cocci. No yeast cells are produced in tissue for this fungus.

• Spherules are at all stages of development- fragmented spherules to well formed with endospores

• Granulomatous inflammation with caseation is usually observed

Development of Cocci spherules from the inhalation of Arthroconidia from nature

Rhinosporidium seeberi (aquatic parasite) forms spherules but much larger than the Cocci spherules - they are usually > 80 uM in size. Also R. seeberi almost always cause oral or nasal mass lesions

Oral or nasal mass lesionsof Rhinosporidium seeberi

Coccidioidesspherules

Coccidioides is not the only spherule forming organism!

Paracoccidioides brasiliensis

• South American Blastomycosis – endemic area Brazil, Venezuela, Columbia

• Inhale infectious particle from soil• >95% of infections in males, possibly due to

estrogen inhibition of mycelial to yeast transformation

• Disease presentation:1. Pneumonia2. Disseminated infection to 2 or more organs3. Extrapulmonary lesions on the face and oral

mucosa

Paracoccidioides

• Cultures at 30*C for mycelial phase are usually not done due to slow growth and nonspecific sporulation

• Culture @ 37˚C• Slow growing yeast• Large yeast (10 – 30uM) with multiple daughter buds (2 – 10

uM) in size • Unique yeast cell known as the Mariner’s wheel or Pilot’s wheel

yeast

Tissue Exam ofParacoccidioides brasiliensis

If more than 2 buds off mother cell – High likelihood it is ParacoccidioidesGranulomatous inflammation with GiantCells

Sporotrichosis

• Sporothrix schenckii• Cutaneous inoculation of fungus from penetrating injury with a

spore or thorn (rose bush)• Initial skin lesion w/wo ulceration• Lymph-cutaneous spread – bone – systemic• Pulmonary and CNS infections are rare but reported

Starts as one ulcerative lesion and then chainsUp the lymphatics – can involve lymph nodes and bone

Sporothrix schenckii•MOLD PHASE

• 30*˚C growth in 3 -5 days• Turns brown to black over time• Septate hyphae with conidia in daisy wheel pattern

•YEAST PHASE• At 37˚C small oval yeast cells, elongated 2 – 5 µM, described as cigar bodies

Sporothrix schenckii• Histology –

• Pyogenic – to – granulomatous inflammation• Hard to find yeast in human tissue• Asteroid body known as Splendore-Hoeppli phenomenon

can be seen, but not specific to Sporothrix, also seen in:• Zygomycetes (Mucorales)• Aspergillus• Blastomycosis• Candida

Daisy like spore arrangement

Sporothrix schenckii

Green colony with red diffusable pigment Uncommon dimorphic fungus The only species of Penicillium that is dimorphicCauses skin lesions in tropics and Pneumonia in immune suppressed

Penicillium marneffei

Penicillium marneffei yeast like cells in tissue

SUBCUTANEOUS FUNGAL INFECTIONSVery unique structures in tissue!

Subcutaneous Fungal Infections Most common will be described• Mycetoma [2 types]

• Actinomycotic – caused by higher bacteria• Eumycotic – caused by dark pigmented molds

• Chromomycosis [Chromoblastomycosis]• Phaeohyphomycosis• Sporotrichosis

Mycetoma• First observed in India and known as Madura Foot or Maduromycosis • Found in the hot temperate parts of the

world

• Three criteria describe Mycetoma:1. Lesions lead to swollen extremities2. Draining sinuses3. Sulfur granules observed in tissue and found in the

weeping drainage

• Fungus grows on organic debris in soil• Implanted into subcutaneous tissue from trauma

Swollen extremity and draining sinus with sulfur granules

Sulfur Granule

Mycetoma

Mycetoma • There are two types of Mycetoma:

1. Actinomycotic mycetoma – caused by higher bacteria species

2. Eumycotic mycetoma – caused by the black molds

• Actinomycotic Mycetoma• 98% of cases of Mycetoma• Nocardia species most common cause• Sulfur granules formed in tissue and the granules vary in

color and contain a matrix of the filamentous bacteria

Gram stain as filamentous Gram positive bacilli – can be poorly staining and appear speckled.Nocardia are positive [red] on the Modified Kinyoun stain.

Modified acid-fast stain[modified Kinyoun stain]

Gram stain of sputum containing Nocardia

Nocardia

Edge of granule has thin filamentous bacteriafor both bacteria – Nocardia is modified acid fast [PAF] positive and is aerobic bacteria. Actinomyces is PAF negative and grows anaerobically.

Beware! Sulfur granule caused by Actinomyces israelii looks identical.

Actinomycotic sulfur granule - Nocardia

Requires 3 – 5 days to grow on agar media [Sabs, 5% Sheep’s blood agar

Colony is dry and crumblyMusty smell

Total of 85 speciesNocardia asteroides is the most common species isolated from human infection

Identification by HPLCor molecular methods

Nocardia species cause mycetoma, and can also cause Pulmonary and Brain infections

Eumycotic mycetoma – subcutaneous infection caused by the black molds

Numerous species of pigmented/black fungi found naturally in the soil can cause this type of infectionCause @ 2% of cases of mycetoma Traumatic implantation injects the mold into the subcutaneous tissueMost common species of black mold include:

Cladophialophora (Cladosporium) carrioniiCladophialophora bantianaPhialophora verrucosaFonsecaea pedrosoiExophiala speciesWangiella species

Eumycotic sulfur granule – the granule is full of a matrix of thick fungal hyphae

Chromomycosis/Chromoblastomycosis

• Three characteristics describe Chromomycosis• Wart like lesions in subcutaneous tissue• Sclerotic bodies observed in tissue• Growth of dark/pigmented fungi

• Black mold naturally found in the soil cause infection through abrasion/ implantation

• Black molds that can cause Chromomycosis:• Cladophialophora [Cladosporium] carrionii• C. bantiana• Phialophora verrucosa• Fonsecaea pedrosoi• Exophiala species• Wangiella species

Chromomycosis/Chromoblastomycosis

Wart like/Verrucous lesionsIn subcutaneous tissue

Sclerotic Body/Medlar Body/Copper Penny is the uniquestructure found in tissue

Prototheca wickerhamii – the cause of Protothecosis• Algae without chlorophyll• Causes skin lesions & nodules• Most common in patients with suppressed immune

system• Compare morula of Protothecosis to sclerotic body of

Chromomycoses

Phaeohyphomycosis This infection is caused by traumatic implantation of dark

fungi into subcutaneous tissue• Variety of infections but nodules/lesions most common

with/without dissemination• Dark hyphae only observed in tissue

Black molds/Dark molds also known as Dematiaceous fungi Black colored colonies and the reverse [back of colony] is also blackNaturally brown hyphae and sporesOne of the major causes of mold growth due to water damage!

Black Molds – Dematiaceous fungi• Black colonies• Brown hyphae and spores• Numerous species • Difficult to identify

• All have one of four types of sporulation• Rhinocladiella-like• Cladosporium-like• Phialophora-like• Acrotheca-like

Rhinocladiella type sporulation

Phialophora type sporulation

Cladophialophora [Cladosporium type sporulation]

Acrotheca type sporulation

Exophiala species

Black Molds that can cause Mycetoma/Chromomycosis/PhaeohyphomycosisThese are difficult to identify but viewing is necessary!

Cladophialophora bantiana

Wangiella dermatitidis

Phialophora verrucosa

AlternariaOther black molds of importance:

Bipolaris australiensis

Very invasive fungal infection:Skin, nasalsinuses, bonebrain

Curvularia lunata

Center cell is the largest

Opportunistic fungal pathogenReported in patients with Bone Marrow Transplantation and Aplastic anemiaCan cause unusual erythematous skin lesions

Exserohilum rostrum• Associated with compounded pharmaceutical [steroid]

products contaminated with dust/dirt• Used for infections into lumbar spine and knee joints for

pain management• Meningitis• Spinal abscess• Synovial infections

Scedosporium apiospermum/Pseudallescheria boydiiCat fur-like colony

IMPORTANT YEAST CAUSING HUMAN INFECTION

Candida speciesCryptococcus neoformans & Cryptococcus gattii

Cutaneous and Superficial Mycoses

• Candida species (@ 10 found in humans)• Opportunistic pathogen involving skin or mucous

membranes from excessive exposure to moisture, antibiotics, or immune suppression

• Yeast is from endogenous source – found as normal flora in the GI and GU tracts and skin

• Variety of infections including: Thrush, vaginitis, skin lesions, nail, diaper rash, to more serious infections like fungemia and endoarditis.

Candida species

• Candida albicans – most common species causing @ 60% of human yeast infections

• Candida glabrata, C. krusei, and C. tropicalis are becoming more common in infection

• These 3 species are more likelyto be resistant to Fluconazole

• Candida parapsilosis has emerged as a pathogen of children and IV lines

Candida species

• Grow in 24 – 48 hours• SABS, IMA, BAP

• Bacteria-like colony – pasty white• Budding yeast – oval @ 7-8 um in size

form pseudohyphae (Yeast do not detach and form elongated hyhae) Can appear like sausage links

• Exception **Candida glabrata is @ 4 µM in size and does NOT form any pseudohyphae

Candida albicansIdentification

• Germ tube formation• Incubate small amount of yeast in serum for 3-4hr at 35 ˚C• Do not incubate >4 hr – this can lead to a false positive

reaction with C. tropicalis• C. dubliniensis also positive (uncommon yeast isolate)

• Chlamydospore formation• Growth on cornmeal agar >48 hrs• Rudimentary structures

C. albicanschlamydospore

C. glabrata only forms yeastNo pseudohyphae

ChromAgar for the identification of Candida

Chromogenic substratesTurn different colors with4 different yeast species

Yeast with pseudohyphae

Candida Histopathology• Pyogenic to granulomatous• Usually observe yeast cells, pseudohyphae and/or hyphae

appearing structures• Candida glabrata = smaller yeast cells and no pseudohyphae

GMS stain of Candida glabrataCandida species not glabrata

Cryptococcus neoformans

• In nature forms a 2um non-encapsulated yeast cell. It is associated with bird droppings (esp. pigeon). C neoformans is enriched by the nitrogen in the droppings.

• Yeast cells are inhaled – travels through the pulmonary system with hematogenous spread to brain and meninges

• Has tropism to the meninges• Infects mostly compromised hosts - AIDS

Cryptococcus neoformans

• Irregular sized (2 – 20uM) yeast cells• Polysaccharide capsule is virulence factor and it’s presence is used in

diagnostic tests for C. neoformans • India ink exam of CSF is a negative staining method/capsule not

stained, • Sensitive test for AIDS patients (90% sensitive)

• Cryptococcal antigen test – capsular polysaccharide is detected in both CSF and serum,• Test for diagnosis and can also follow recovery with falling titer /more

sensitive than India ink• Grows on mycologic agars but is sensitive to cycloheximide –

• Mucoid colonies due to capsule polysaccharide formation• Urease enzyme + Inositol assimilation +• Brown colonies produced on bird seed agar

Cryptococcus gattii – a closely related relative of C. neoformans• Isolated from forested area of the Pacific Northwest (British Columbia, Washington, Oregon and California) found in soil debris and tree species• Infection of normal and immune suppressed hosts• Mostly Pulmonary disease [Cryptococcoma] but can develop meningitis like C. neoformans• Culture and staining identical to C. neoformans except for L Canavanine glycine bromthymol blue medium –

C. gatti = blue

C. neoformans = colorless

Positive India Ink

Urea medium demonstrating urease enzyme activity of Cryptococcus

Observe Budding cells

Variability in size

Positive

Mucoid colonies ofC. neoformans andC. gattii

C. neoformans/C. gattii formsbrown colonies on Birdseed agar

Mucicarmine stain

Stains the capsular polysaccharide of capsule

Pneumocystis jiroveci could be confused with C. neoformans – Careful! Central nuclear staining

C. neoformans/ C. gattii

CUTANEOUS AND SUPERFICIAL MYCOSESMalassezia furfurDermatophytes

Malassezia furfur

• Pityriasis versicolor• Most superficial of the dermatomycoses • Found as normal flora on the skin,• More common on oily skin or high use of skin oils• Diseases: • Skin: macules, papules, patches, plaques on chest back and shoulders with either hypo or hyper pigmentation – does not invade into deeper tissues

• Fungemia in neonates caused by skin flora tunneling in the IV lipid feeding lines

Malassezia furfur• Lipophilic yeast – oil required for growth

• Media used for culture must contain oil or have oil overlay

• Small budding yeast 2 – 4 µM with collarette

Spaghetti and meatballs

Size range for Yeast• Candida glabrata/Histoplasma capsulatum

• 2 – 4 um• Candida species

• 8 – 10 um plus pseudohyphae• Cryptococcus neoformans/gattii

• 2 – 20 um• Blastomyces dermatitidis

• 8-15 um

Dermatophytes – Ringworm infections

• Hair, skin and nail infections• 3 genera of fungi

• Microsporum species (many)• Epidermophyton floccosum• Trichophyton species (many)

• Disease described by area of the body infected: tinea capitis (head), t. pedis (foot)

• Usually a clinical diagnosis not requiring culture• Can do a KOH prep or Calcofluor white prep to visualize fungal hyphae

Positive KOH prepShowing thin septate fungal hyphae

Calcofluor white stain with fluorescence – thin fungal hyphae

Microsporum canisMain cause of ringworm from dog and catWhite colony/ yellow on backside of colonyTuberculate macroconidia [spiny projections]Few if any microconidia

Microsporum gypseum infection from exposure to contaminated soil

Trichophytonrubrum

White colony with red diffusable pigment

Pencil shapedMacroconidiaMany micro-Conidia

Infection from fomites

Red diffusible pigment

Trichophytontonsurans

Ballooning Microconidia

Primary cause of epidemic scalp ringworm in children from infection of Hair

Epidermophyton floccosum

Beaver tail large spores without microconidiaKhaki green colony

Opportunistic Fungal PathogensInfections in the immune suppressed host

or special circumstancesHyaline moldsBlack molds

Opportunistic Fungi - hyaline

• Hyaline – no color to the hyphae• Regular septations in the hyphae • Branching – angle can be helpful in identification• Usually grow in 3 – 5 days at 30˚C• ??? of species thousands– taxonomy changing daily

Aspergillus species• Hyaline with septations• Numerous round conidia• In tissue - Branching at 45 degree angle• Primarily pulmonary infection in immune suppressed• Invade vessels, cause thrombosis & infarctions

Aspergillus Structure

Aspergillus speciesFour species most common in human infections:1. Aspergillus fumigatus 2. Aspergillus flavus3. Aspergillus niger4. Aspergillus terreus – unique and important – only

Aspergillus species resistant to Amphotericin B

Aspergillus Galactomannan Enzyme immunoassay – detects circulating Aspergillus antigen in the blood and/or bronchial lavage fluid

Problems with low sensitivity and specificity False positive reaction in patients on therapy with Piperacillin/Tazobactam, infected with Histoplasma capsulatum

Aspergillus fumigatus

Blue/Green colonyPhialids with spores areDirected upward

Aspergillus flavus

Green colonyOrange colored spores that surround the vesecle

Aspergillus niger

Black colonyBlack spores surround the vesicle

Aspergillus terreus Sandy colored colony

Aleurioconidia

Resistance to Amphotericin B

Aspergillus•fruiting head dichotomous•branching septate hyphae• branching at 45* angle

Pseudallescheria boydiican be confused for Aspergillus, hyphae is a bit thinner. Culture separates the two fungi.

Fusarium species – Common in nature/plantsdisease related to immune status of hostInfections reported: Disseminated in bone marrow

transplantsCorneal infections in contact

lens wearers

Scopulariopsis species –Soil and plantsInfections: Nail, skin, sinusitis, pulmonary and disseminated

Paecilomyces speciesIsolated from soil and foodOpportunistic pathogen in the immune suppressed

Penicillium species – most common mold in the environment, bread mold, uncommon cause of human disease

MUCORMYCOSIS/ZYGOMYCOSISHyalineBroad non-septate hyphae

Mucormycosis/Zygomycosis• Infections in diabetics, the elevated glucose enriches fungal growth – classic infection is rhinocerebral mucormycosis

• Sinus and pulmonary infection in the immune suppressed host

• Broad, hyaline, aseptate hyphae produced• Cultures grow in 24 hrs, coarse aerial hyphae• Can be difficult to culture – tube like hyphae killed during manipulation and plating

• Should not grind tissue• Mince tissue and place on agar

Zygomycete – coarse, aerial hyphae after 24 hours growth on SABS agar at 30˚C

Rhizopus Absidia

Distant rhizoids

Mucor

No rhizoids

Rhizoids

90˚ angle branching, aseptate, ribbon like

Invades vessels and can cause infarcts and thrombi

Zygomycetes (Mucorales)